Methods and kits for the determination of the presence and quantity of vitamin d analogs in samples

Abstract

Embodiments of the present invention are directed to methods and kits for determining the presence or absence, and the amount if present, of vitamin D analogs in samples. One embodiment of the present method for detecting the presence or absence of vitamin D analogs in a sample comprises the steps of adding an effective amount of a multiple charge cationic agent to the sample to form a cationic treated sample. The effective amount of a multiple charge cationic agent enhances the signal from vitamin D analogs upon analysis by mass spectroscopy.

1 . A method for detecting the presence or absence of Vitamin D analogs in a sample comprising the steps of:
 adding an effective amount of a multiple charge cationic agent to said sample to form a cationic treated sample, said effective amount of said multiple charge cationic agent enhancing a signal from Vitamin D analogs upon analysis by mass spectroscopy;   subjecting said cationic treated sample to centrifugation to form a supernatant;   separating proteins in said supernatant from said Vitamin D analogs by solid phase extraction to form an analyte solution;   placing said analyte solution on an analytical column and forming a eluent having said Vitamin D analogs separated from non Vitamin D compounds,   placing said eluent in a mass spectrometer and detecting the presence or absence of Vitamin D analogs in the sample by mass spectra.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein said solid phase extraction is performed by loading said supernatant on a chromatographic media and eluting said Vitamin D analogs from said chromatographic media to form said analyte solution, said analyte solution having Vitamin D analogs in the event said Vitamin D analogs are present in said sample. 
     
     
         4 - 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein said sample is serum. 
     
     
         8 . The method of  claim 1 , wherein said multiple charge cationic agent is selected from the group consisting of metal sulfate salts. 
     
     
         9 . The method of  claim 1 , wherein said metal sulfate salt is zinc sulfate. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , further comprising adding a known amount of a labeled Vitamin D analog to said sample. 
     
     
         12 . The method of  claim 11 , wherein said labeled Vitamin D analog is selected from the group consisting of deuterated 25(OH) Vitamin D3 and deuterated 25(OH) Vitamin D2. 
     
     
         13 . The method of  claim 1 , wherein said analyte solution is dried and reconstituted in methanol and water prior to placing said analyte solution on the said analytical column. 
     
     
         14 . (canceled) 
     
     
         15 . A kit for detecting the presence or absence of Vitamin D analogs in a sample comprising:
 an effective amount of a multiple charge cationic agent, a preparatory chromatographic media for separating the Vitamin D analogs from each non-vitamin D compositions, and instructions for use of said multiple charge cationic agent in a method to detect the presence or absence of Vitamin D analogs;   said method comprising adding said effective amount of a multiple charge cationic agent to said sample to form a cationic treated sample, said effective amount of the multiple charge cationic agent enhancing the signal from Vitamin D analogs upon further analysis by mass spectroscopy; centrifuging said treated sample to form a supernatant, separating protein in said supernatant from said Vitamin D analogs by protein precipitation with said protein precipitating agent to form an analyte solution, placing said analyte solution on an analytical column and forming an eluent having said Vitamin D analogs separated from non Vitamin D compounds, and placing said eluent in a mass spectrometer and detecting the presence or absence of Vitamin D analogs in the sample by mass spectra.   
     
     
         16 - 21 . (canceled) 
     
     
         22 . The kit of  claim 15 , wherein said multiple charge cationic agent is selected from the group consisting of metal sulfate salts. 
     
     
         23 . The kit of  claim 15 , wherein said metal sulfate salt is zinc sulfate. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The kit of  claim 15 , wherein said kit further comprises a known amount of a labeled Vitamin D analog to facilitate quantification by mass spectroscopy. 
     
     
         27 . The kit of  claim 26 , wherein said labeled Vitamin D analog is selected from the group consisting of deuterated 25(OH) Vitamin D3 and deuterated 25(OH) Vitamin D2. 
     
     
         28 . The kit of  claim 15 , further comprising an analytical column. 
     
     
         29 . The kit of  claim 15 , wherein said analytical column has a chromatographic media comprising a bridged ethyl hybrid material. 
     
     
         30 . The kit of  claim 15 , wherein the preparatory chromatographic media comprises a polymeric media with a hydrophilic and a hydrophobic component.